Functionally modified polypeptides and radiobiosynthesis

ABSTRACT

Provided herein are compositions and methods for generating polypeptides using non-natural amino acids (nnAAs) and genetic machinery, wherein the modified polypeptides, such as therapeutic polypeptides, bind to albumin, such as serum albumin. Methods of substituting a non-natural amino acid in a first polypeptide to obtain a modified polypeptide, the nnAA in some instances comprising an albumin targeting group, are disclosed, as are methods for making populations of such modified polypeptides. A therapeutic polypeptide, interleukin-1 receptor antagonist (IL-1RA) is exemplified using the disclosed methods.

RELATED APPLICATIONS

This patent application claims the benefit of U.S. Provisional PatentApplication No. 62/383,382, filed Sep. 2, 2016, which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The invention is directed to the field of protein drugs. Specifically,the invention is directed to improving the pharmacokinetics of proteindrugs through the incorporation of non-natural amino acids intotherapeutic polypeptides using genetic machinery.

BACKGROUND

More than one hundred therapeutic proteins are used currently to treatcancer, immune diseases, and diabetes, among other disorders, andhundreds more are in clinical trials (Dimitrov D S. TherapeuticProteins. In: Therapeutic Proteins: Methods and Protocols, Methods inMolecular Biology, Editor: Voynov V, Caravella J A. SpringerScience+Business Media; 2012. p. 1-26). Antibodies comprise half of themarket of currently prescribed therapeutic peptides. In part, theutility of antibodies derives from their relatively long serumhalf-lives compared to other peptides and proteins, which are generallycleared and/or metabolized quickly. Such rapid clearance often dictatesaggressive dosing schedules for non-antibody therapeutic proteins, whichcan increase the risk of side effects, shrink the therapeutic window,and have a negative impact upon patient compliance. For example,Kineret® (anakinra), a 17 kD therapeutic protein currently prescribedfor the treatment of rheumatoid arthritis, is administered by dailysubcutaneous injection (70-100 mg, 1-2 mg/kg) over several months. Peakanakinra serum concentrations occur at ˜6 h after injection.

Several strategies are currently employed to extend the serum lifetimeof therapeutic peptides and proteins (Pollaro L, Heinis C. Strategies toprolong the plasma residence time of peptide drugs. MedChemComm. 2010;1, 319-24), including alteration of peptide sequence and secondarystructure to minimize protease activity (Timmerman P et al. Functionalreconstruction and synthetic mimicry of a conformational epitope usingCLIPS™ technology. J Mol Recognit. 2007; 20, 283-99; Houston M E et al.Lactam Bridge Stabilization of α-Helices: The Role of Hydrophobicity inControlling Dimeric versus Monomeric α-Helices. Biochemistry (Mosc).1996; 35, 10041-50; Sim S, et al. Directional Assembly of α-HelicalPeptides Induced by Cyclization. J Am Chem Soc. 2012; 134, 20270-2) andPEGylation (Veronese F M. Peptide and protein PEGylation: a review ofproblems and solutions. Biomaterials. 2001; 22, 405-17; Jevševar S, etal. PEGylation of therapeutic proteins. Biotechnology Journal. 2010; 5,113-28; Greenwald R B, et al. Effective drug delivery by PEGylated drugconjugates. Advanced Drug Delivery Reviews. 2003; 55, 217-50; Xue X, etal Phenyl Linker-Induced Dense PEG Conformation Improves the Efficacy ofC-Terminally MonoPEGylated Staphylokinase. Biomacromolecules. 2013; 14,331-41) to limit globular filtration. A more promising, and potentiallygeneralizable, approach is to introduce human serum albumin (HSA)targeting elements into the protein (Kratz F. Albumin as a drug carrier:Design of prodrugs, drug conjugates and nanoparticles. J ControlledRelease. 2008; 132, 171-83; Dennis M S, et al. Albumin Binding as aGeneral Strategy for Improving the Pharmacokinetics of Proteins. J BiolChem. 2002; 277, 35035-43). Albumin targeting is currently accomplishedeither by genetic engineering or post-translational bioconjugationstrategies. Genetically encoded albumin-targeting peptides (AlbudAb™(O'Connor-Semmes R L, et al. GSK2374697, a Novel Albumin-Binding DomainAntibody (AlbudAb), Extends Systemic Exposure of Exendin-4: First Studyin Humans-PK/PD and Safety. Clin Pharmacol Ther (N Y, N.Y., U S). 2014;96, 704-12; WO2010108937A2; Holt Li, et al. Anti-serum albumin domainantibodies for extending the half-lives of short lived drugs. ProteinEng, Des Sel. 2008; 21, 283-8; Holt U, et al. Domain antibodies:proteins for therapy. Trends Biotechnol. 2003; 21, 484-90) and others(Dennis, 2002, supra; Langenheim J F, Chen W Y. Improving thepharmacokinetics/pharmacodynamics of prolactin, GH, and theirantagonists by fusion to a synthetic albumin-binding peptide. JEndocrinol. 2009; 203, 375-87) can be incorporated in a highlycontrolled fashion to extend the serum lifetime of therapeutic peptides,but this improvement in pharmacokinetic properties (PK) comes at thecost of a relatively large perturbation to the native protein structure.In contrast, post-translational modification of proteins can introducerelatively small albumin targeting molecules, but this approach canyield heterogeneous mixtures that are often difficult to characterizefully, and include macromolecular species with potentially widelyvarying PK and target affinity.

Significant progress toward the control of post-translationalmodification is possible with the introduction of genetically encoded“clickable” amino acids (Kurra Y, et al. Two Rapid Catalyst-Free ClickReactions for In Vivo Protein Labeling of Genetically Encoded StrainedAlkene/Alkyne Functionalities. Bioconjugate Chem. 2014; 25, 1730-8;Milles S, et al. Click Strategies for Single-Molecule ProteinFluorescence. J Am Chem Soc. 2012; 134, 5187-95; Hertweck C.Biosynthesis and Charging of Pyrrolysine, the 22nd Genetically EncodedAmino Acid. Angew Chem, Int Ed. 2011; 50, 9540-1; Plass T, et al.Genetically Encoded Copper-Free Click Chemistry. Angew Chem, Int Ed.2011; 50, 3878-81, S/1-S/13; Nguyen D P, et al. Genetic encoding andlabeling of aliphatic azides and alkynes in recombinant proteins via apyrrolysyl-tRNA Synthetase/tRNA(CUA) pair and click chemistry. J Am ChemSoc. 2009; 131, 8720-1), but this approach has inherent weaknesses fortherapeutic protein development: the chemistry used to “click” on thealbumin targeting group needs to be extremely efficient, and methods arerequired to separate the functionalized protein from those featuringunreacted or partially degraded side chains. Despite vast improvementsin bioconjugation using click chemistry, generalizable reagents andreactions conditions required to meet these criteria are still lacking(Reddington S C, et al. Residue choice defines efficiency and influenceof bioorthogonal protein modification via genetically encoded strainpromoted Click chemistry. Chem Commun (Cambridge, U K). 2012; 48,8419-21).

An ideal solution to the albumin-targeting problem for therapeuticproteins would maintain the control of the genetic approach, cause aminimal structural perturbation characteristic of a small moleculealbumin targeting tag, and not require any post-translational proteinmodification.

SUMMARY OF INVENTION

In a first aspect, disclosed herein are methods of substituting anatural amino acid in a first polypeptide with a non-natural amino acidto obtain a modified polypeptide, wherein the non-natural amino acidcomprises a moiety selected from the group consisting of: analbumin-targeting group, a radiolabel, and a radiolabelledalbumin-targeting group, comprising:

-   -   (a) selecting at least one amino acid residue in the first        polypeptide to be substituted with the non-natural amino acid;    -   (b) selecting a polynucleotide encoding the first polypeptide;    -   (c) modifying the polynucleotide such that the amino acid        residue to be substituted is encoded by a nonsense codon;    -   (d) expressing the modified polynucleotide in a cell, wherein        the cell is in the presence of the non-natural amino acid and        expresses a suppressor tRNA and its cognate tRNA synthetase        wherein the suppressor tRNA recognizes the nonsense codon of        step (c), and the cell incorporates the non-natural amino acid        into the modified polypeptide at the nonsense codon of step (c)        during translation. Optionally, a further purifying step (e) can        be included. In such aspect, the non-natural amino acid can be        represented by the formula:        A-T

wherein A is an amino acid selected from the group consisting of lysine,ornithine, arginine, serine, threonine, asparagine and glutamine, and

T is a moiety selected from the group consisting of: analbumin-targeting group, a radiolabel, and a radiolabelledalbumin-targeting group. In one embodiment, T is an albumin targetinggroup. In an embodiment, the albumin targeting group is selected fromthe group consisting of:

In another embodiment, the albumin targeting group comprises an aryl orheteroaryl moiety and a thioyl moiety, such that A-T comprises athioamide moiety. In a particular embodiment, the thioyl moiety isconjugated to the aryl or heteroaryl moiety.

The non-natural amino acid can be Nε-(4-(4-iodophenyl)butanoyl)lysine,NE-(4-(4-iodophenyl)butanethioyl)lysine, or pharmaceutically acceptablesalts thereof. The non-natural amino acid can comprise a radiolabelledalbumin-targeting group. The non-natural amino acid can comprise aradiolabel. The nonsense codon can be an amber, opal, or ochre nonsensecodon. The non-natural amino acid can be radiolabelled, such as with¹²³I, ¹²⁴I, ¹²⁵I or ¹³¹I. The suppressor tRNA can be tRNA_(CUA) ^(Pyl)with its cognate tRNA synthetase can be pyrrolysyl-tRNA synthetase. Thecell can be radiation resistant. The cell can be a bacterial cell, afungal cell, a plant cell, or a mammalian cell, such as a bacterialEscherichia coli cell or a fungal Saccharomyces cerevisiae cell. In thecase of a radiation-resistant bacterial cell, the RecA, YfjK, and DnaBproteins in the cell can be mutated (comprising the mutations for eachprotein RecA (D276N); YfjK (A151D); and DnaB (P80H)); and in the case ofa radiation-resistant E. coli cell can be derived from E. coli strainC321.ΔA. In the case where the cell is a bacterial cell, the cell cancomprise only nonsense codons that are not the nonsense codon present inthe polynucleotide and/or does not express factors that decode thenonsense codon present in the polynucleotide; for example, the nonsensecodon present in the nucleotide is an amber nonsense codon, and thefactor is release factor-1. In this first aspect, the first polypeptidecan be a therapeutic polypeptide, such as one selected from the groupconsisting of anticoagulants, blood factors, bone morphogeneticproteins, engineered protein scaffolds, enzymes, growth factors,hormones, interferons, interleukins, and thrombolytics. Alternatively,the therapeutic polypeptide is an antigen-binding polypeptide, such asone selected from the group consisting of an antibody, a chimericantibody, a monoclonal antibody, a single chain antibody, Fab, Fab′,F(ab′)₂, Fv, and scF. The therapeutic polypeptide can be an Fc fusion.The therapeutic polypeptide can be an interleukin-1 receptor antagonist.In this first aspect, the modified polypeptide can have enhanced bindingto human serum albumin when compared to the first polypeptide and/or canhave enhanced pharmacokinetic properties when compared to the firstpolypeptide. In the case of enhanced pharmacokinetic properties, suchproperties can be increased serum half-life.

In a second aspect, disclosed herein are cells comprising a modifiedpolynucleotide encoding a modified polypeptide, a suppressor tRNA, and acognate tRNA synthetase,

wherein the modified polynucleotide comprises one or more nonsensecodons recognized by the suppressor tRNA, and the cognate tRNAsynthetase,

wherein the modified polypeptide comprises one or more non-natural aminoacids at positions encoded by the nonsense codons, and

wherein the non-natural amino acids comprise a moiety selected from thegroup consisting of: an albumin-targeting group, a radiolabel, and aradiolabelled albumin-targeting group. The nonsense codon can be anamber, opal, or ochre nonsense codon. The non-natural amino acid cancomprise an albumin targeting group, and can be, for example,Nε-(4-(4-iodophenyl)butanoyl)lysine,Nε-(4-(4-iodophenyl)butanethioyl)lysine, or pharmaceutically acceptablesalts thereof. The non-natural amino acid can comprise a radiolabel,such as ¹²³I, ¹²⁴I, ¹²⁵I or ¹³¹I. In one embodiment, the non-naturalamino acid is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.

The non-natural amino acid can comprise a radiolabel, such as ¹²³I,¹²⁴I, ¹²⁵I or ¹³¹I. The non-natural amino acid can also be selected fromthe group consisting of:

and pharmaceutically acceptable salts thereof.

In this second aspect, the tRNA can be tRNA_(CUA) ^(Pyl) and its cognatetRNA synthetase is pyrrolysyl-tRNA synthetase. The cell can be radiationresistant, and can be a bacterial cell, a fungal cell, a plant cell, ora mammalian cell, such as a bacterial Escherichia coli cell or a fungalSaccharomyces cerevisiae cell. In the case of a bacterial cell, theRecA, YfjK, and DnaB proteins in the cell can be mutated. The E. colicell can be derived from E. coli strain C321.ΔA. In the case of an E.coli bacterial cell, the mutations can be RecA (D276N); YfjK (A151D);and DnaB (P80H). In the case of a bacterial cell, the cell can compriseonly nonsense codons that are not the nonsense codon present in thepolynucleotide and/or not express factors that decode the nonsense codonpresent in the polynucleotide; for example, the cell can comprise anonsense codon that is an amber nonsense codon, and the factor isrelease factor-1. In such second aspect, the first polypeptide can be atherapeutic polypeptide, such as one selected from the group consistingof anticoagulants, blood factors, bone morphogenetic proteins,engineered protein scaffolds, enzymes, growth factors, hormones,interferons, interleukins, and thrombolytics. The therapeuticpolypeptide can be an antigen-binding polypeptide, such as one selectedfrom the group consisting of an antibody, a chimeric antibody, amonoclonal antibody, a single chain antibody, Fab, Fab′, F(a′)₂, Fv, andscF. In other sub-aspects, the therapeutic polypeptide is an Fc fusion.The therapeutic polypeptide can be an interleukin-1 receptor antagonist.In this second aspect, the non-natural amino acid can comprise analbumin-targeting group. The modified polypeptide of this second aspectcan have enhanced binding to human serum albumin when compared to thefirst polypeptide; furthermore, the modified polypeptide can haveenhanced pharmacokinetic properties when compared to the firstpolypeptide, such as increased serum half-life.

In a third aspect, disclosed herein are methods of making a modifiedpolypeptide that binds to albumin, comprising:

(a) selecting at least one amino acid residue in a first polypeptide tobe substituted with a non-natural amino acid disclosed herein;

(b) selecting a polynucleotide encoding the polypeptide;

(c) modifying the polynucleotide such that the amino acid residue to besubstituted is encoded by a nonsense codon;

(d) expressing the modified polynucleotide in a cell, wherein the cellis in the presence of the non-natural amino acid, and expresses asuppressor tRNA and its cognate tRNA synthetase, wherein the suppressortRNA recognizes the nonsense codon of step (c), and the cellincorporates the non-natural amino acid into a modified polypeptide atthe nonsense codon of step (c) during translation. The method canfurther comprise a set (e), purifying the modified polypeptide. Thenonsense codon can be an amber, opal, or ochre nonsense codon. Thenon-natural amino acid can be radiolabelled, such as with ¹²³I, ¹²⁴I,¹²⁵I or ¹³¹I. The suppressor tRNA can be tRNA_(CUA) ^(Pyl) and itscognate tRNA synthetase is pyrrolysyl-tRNA synthetase. The cell can beradiation resistant, and can be a bacterial cell, a fungal cell, a plantcell, or a mammalian cell. The cell can be a bacterial Escherichia colicell, or a fungal Saccharomyces cerevisiae cell. In the case of aradiation-resistant bacterial cell, the RecA, YfjK, and DnaB proteins inthe cell can be mutated, including the mutations RecA (D276N); YfjK(A151D); and DnaB (P80H). The bacterial cell can be derived from E. colistrain C321.ΔA. In the case of a bacterial cell, the cell can compriseonly nonsense codons that are not the nonsense codon present in thepolynucleotide. The bacterial cell can also not express factors thatdecode the nonsense codon present in the polynucleotide; for example,the nonsense codon present in the nucleotide can be an amber nonsensecodon, and the factor can be release factor-1. The first polypeptide canbe a therapeutic polypeptide, such as one selected from the groupconsisting of anticoagulants, blood factors, bone morphogeneticproteins, engineered protein scaffolds, enzymes, growth factors,hormones, interferons, interleukins, and thrombolytics. The therapeuticpolypeptide can also be an antigen-binding polypeptide, such as oneselected from the group consisting of an antibody, a chimeric antibody,a monoclonal antibody, a single chain antibody, Fab, Fab′, F(ab′)₂, Fv,and scF. The therapeutic polypeptide can be a Fc fusion. The therapeuticpolypeptide can be an interleukin-1 receptor antagonist. In such thirdaspect, the modified polypeptide can have enhanced binding to humanserum albumin when compared to the first polypeptide. Furthermore, themodified polypeptide can have enhanced pharmacokinetic properties whencompared to the unmodified polypeptide, including increased serumhalf-life.

In a fourth aspect, disclosed herein are methods of engineering anEscherichia coli cell to be resistant to radiation, wherein the cell isused to introduce radiolabeled non-natural amino acids intopolypeptides, comprising mutating one or more proteins in the cell. Themutated proteins can comprise RecA, YfjK, and DnaB proteins, and canhave mutations RecA (D276N); YfjK (A151D); and DnaB (P80H). Theradiolabeled amino acids can be labeled with, for example, ¹²³I, ¹²⁴I,¹²⁵I or ¹³¹I. In such fourth aspect, being resistant to radiationcomprises increased survival of the engineered cell compared to a samecell that is not engineered.

In a fifth aspect, disclosed herein are cells, comprising apolynucleotide encoding a first polypeptide that when expressed by thecell is substituted with a non-natural amino acid comprising an albumintargeting group at a nonsense codon in the polynucleotide, creating amodified second polypeptide,

wherein:

the cell comprises a suppressor tRNA and its cognate tRNA synthetasethat recognize the nonsense codon;

the non-natural amino acid is introduced into the first polypeptide by anonsense codon;

the non-natural amino acid is exogenously supplied; and

the modified second polypeptide has enhanced binding to human serumalbumin when compared to the first polypeptide. In one embodiment, thealbumin targeting group is selected from the group consisting of:

In another embodiment, the albumin targeting group comprises an aryl orheteroaryl moiety and a thioyl moiety, such that the non-natural aminoacid comprises a thioamide moiety. In a particular embodiment, thethioyl moiety is conjugated to the aryl or heteroaryl moiety.

The nonsense codon can be an amber, opal, or ochre nonsense codon. Thecells can further lack a factor that decodes the nonsense codonrecognized by the tRNA. The non-natural amino acid can beNε-(4-(4-iodophenyl)butanoyl)lysine, and the non-natural amino acid cancomprise a radiolabel, such as in the case ofNε-(4-(4-iodophenyl)butanoyl)lysine, the radio label can be ¹²³I, ¹²⁴I,¹²⁵I or ¹³¹I. The cell can be radiation resistant, and can be abacterial cell, a fungal cell, a plant cell, or a mammalian cell. Thecell can be a bacterial Escherichia coli cell, or a fungal Saccharomycescerevisiae cell. In the case of a radiation-resistant bacterial cell,the RecA, YfjK, and DnaB proteins in the cell can be mutated, includingthe mutations RecA (D276N); YfjK (A151D); and DnaB (P80H). The bacterialcell can be derived from E. coli strain C321.ΔA. In the case of abacterial cell, the cell can comprise only nonsense codons that are notthe nonsense codon present in the polynucleotide. The bacterial cell canalso not express factors that decode the nonsense codon present in thepolynucleotide; for example, the nonsense codon present in thenucleotide can be an amber nonsense codon, and the factor can be releasefactor-1.

In a sixth aspect, disclosed herein is a modified interleukin-1 receptorantagonist polypeptide comprising at least one non-natural amino acid,wherein the non-natural amino acid comprises a moiety selected from thegroup consisting of: an albumin-targeting group, a radiolabel, and aradiolabelled albumin-targeting group. The modified interleukin-1receptor antagonist can be human having a polypeptide sequence of SEQ IDNO:1, and the non-natural amino acid is located at position 71, 93, or135 of the polypeptide of SEQ ID NO:1. The non-natural amino acid can beradiolabelled, such as with ¹²³I, ¹²⁴I, ¹²⁵I or ¹³¹I. The modifiedinterleukin-1 receptor antagonist polypeptide can have enhanced bindingto human serum albumin when compared to an unmodified interleukin-1receptor antagonist polypeptide; it may also have pharmacokineticproperty can comprise enhanced serum half-life.

In a seventh aspect, disclosed herein are methods of making a modifiedpolypeptide comprising

(a) selecting at least one amino acid residue in a first polypeptide tobe substituted with a non-natural amino acid, wherein the substitutedamino acid is solvent-accessible;

(b) selecting a polynucleotide encoding the first polypeptide;

(c) modifying the polynucleotide such that the amino acid residue to besubstituted is encoded by a nonsense codon; and

(d) expressing the modified polynucleotide in a cell, wherein the cellis in the presence of the non-natural amino acid and expresses asuppressor tRNA and its cognate tRNA synthetase wherein the suppressortRNA recognizes the nonsense codon of step (c), and the cellincorporates the non-natural amino acid into a modified polypeptide atthe nonsense codon of step (c) during translation. The method canfurther comprise a set (e), purifying the modified polypeptide. Thenon-natural amino acid can comprise a moiety selected from the groupconsisting of: an albumin-targeting group, a radiolabel, and aradiolabelled albumin-targeting group. The suppressor tRNA can betRNA_(CUA) ^(Pyl) and its cognate tRNA synthetase is pyrrolysyl-tRNAsynthetase.

In an eighth aspect, disclosed herein are compositions comprising aplurality of identical polypeptides,

wherein each polypeptide comprises one or more non-natural amino acids,and

wherein the non-natural amino acids comprise a moiety selected from thegroup consisting of: an albumin-targeting group, a radiolabel, and aradiolabelled albumin-targeting group. In such compositions, thenon-natural amino acids can comprise an albumin-targeting group. The oneor more non-natural amino acids can beNε-(4-(4-iodophenyl)butanoyl)lysine. The non-natural amino acids cancomprise a radiolabel, such as ¹²³I, ¹²⁴I, ¹²⁵I or ¹³¹I.

In a ninth aspect, disclosed herein is a non-natural amino acid compoundcomprising lysine or ornithine and an albumin targeting group, whereinthe lysine or ornithine is linked to the albumin-targeting group by athioamide moiety (i.e., a thioamide linkage). In certain embodiments,the thioamide linkages are more stable to in vivo hydrolysis, ascompared to amide linkages. In certain embodiments, the thioamidelinkages are more stable to peptidase activity, as compared to amidelinkages. In certain embodiments, the non-natural amino acid compoundhas greater in vivo stability than a corresponding compound wherein alysine or ornithine is linked to an albumin-targeting group by an amidemoiety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows non-natural amino acid syntheses (top) and radiosyntheses(bottom) of compounds disclosed herein.

FIG. 2 shows amber suppression in green fluorescent protein (GFP) by aPylRS mutant in the presence of the non-natural amino acidNε-(4-(4-iodophenyl)butanoyl)lysine. Shown from left to right is afluorescence intensity histogram for control, mutant nnAA 1, and mutantnnAA2.

FIG. 3 shows complex of the interleukin-1 receptor (IL-1R) with theinterleukin-1 receptor antagonist (IL-1RA). The sites for substitutionwith 1 and 2 (see FIG. 1) are shown.

FIG. 4 shows dipeptide non-natural amino acids (nnAAs) for HPLC HSAbinding assay studies (see Examples).

FIG. 5 shows a proposed one-step radioiodination route to radiolabelednnAAs (top) based upon one-step synthesis of mIBG from a diaryliodoniumsalt precursor (bottom).

DETAILED DESCRIPTION

Disclosed herein are flexible methods to engineer plasma protein bindingcapability into polypeptides that maintain the advantages of geneticcontrol, but causes a structural perturbation that is commensurate withthat of a small molecule tag. By expanding the genetic code to permitintroduction of an albumin-targeting amino acid, such asNε-(4-(4-iodophenyl)butanoyl)lysine, directly into therapeutic proteinsmeets these requirements. Further disclosed is a simple “toolkit”comprising (1) albumin-targeting, non-natural amino-acids (e.g., lysineor ornithine derivatives); (2) the genetic machinery and modifiedorganisms to introduce this amino acid into therapeutic proteins, and(3) true iodinated radiotracers of therapeutic proteins that permitdirect assessment of the impact of the albumin-targeting groups in vivo,where the term “true radiotracer” is defined as a compound which differsfrom the parent protein only by isotopic substitution of iodine.

Definitions

An “engineered” cell is a cell that differs in at least one propertyfrom a parent cell. These differences can come about due to changes inthe genetic material and/or content of the cell. In some cases, thischange in genetic material is through the introduction of exogenousgenetic material, such as is possible through recombinant procedures.

“Enhanced binding” means the binding between at least two molecules,wherein at least one molecule is changed from its native state so thatthe binding affinity is greater between the two molecules. For example,molecule A may not bind or weakly bind to molecule B, but when moleculeA is modified (A′), such as by the introduction of a non-natural aminoacid having an affinity tag added thereto, molecule A′ binds withgreater affinity for molecule B. In the methods and compositions of theinvention, molecule A′ is a polypeptide modified with a non-naturalamino acid with an albumin-binding tag, such asNε-(4-(4-iodophenyl)butanoyl)lysine, and molecule B is albumin, such ashuman serum albumin. Enhanced binding can be measured using a variety oftechniques, including affinity determination by surface plasmonresonance and direct binding assays

“Enhanced pharmacokinetic properties” means that a modified molecule,when compared to the native molecule, differs in at least onepharmacokinetic property. Pharmacokinetic metrics include dose, dosinginterval, plasma concentration after administration and the time toreach such; concentration, elimination half-life, elimination rateconstant, infusion rate, clearance, bioavailability, fluctuation, etc.For example, an enhanced pharmacokinetic property may include longerserum half-life.

“Expression system” means a host cell, or cellular components andcompatible vector under suitable conditions, e.g., for the expression ofa protein coded for by foreign DNA carried by the vector and introducedto the host cell. Common expression systems include E. coli host cellsand plasmid vectors, insect host cells such as Sf9, Hi5 or S2 cells andBaculovirus vectors, and mammalian host cells and vectors. Otherexpression systems make use of fungal host cells (such as Saccharomycescerevisiae) and plant host cells.

Certain embodiments disclosed herein expressly utilize only a cell-freeexpression or translation system and not a host cell. Certain otherembodiments expressly utilize only an auxotrophic host cell. Stillcertain other embodiments expressly utilize only a non-auxotrophic hostcell, or a prototrophic host cell.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state.

The term “subject” refers to a mammal. A subject therefore refers to,for example, dogs, cats, horses, cows, pigs, guinea pigs, and the like.Preferably the subject is a human. When the subject is a human, thesubject may be referred to herein as a patient.

Various methodologies of the instant invention include steps thatinvolve comparing a value, level, feature, characteristic, property,etc. to a “suitable control”, referred to interchangeably herein as an“appropriate control”. A “suitable control” or “appropriate control” isany control or standard familiar to one of ordinary skill in the artuseful for comparison purposes. In one embodiment, a “suitable control”or “appropriate control” is a value, level, feature, characteristic,property, etc. determined prior to performing a methodology, asdescribed herein. For example, a transcription rate, mRNA level,translation rate, protein level, biological activity, cellularcharacteristic or property, genotype, phenotype, etc. can be determinedprior to performing a methodology. In another embodiment, a “suitablecontrol” or “appropriate control” is a value, level, feature,characteristic, property, etc. determined in a cell or organism, e.g., acontrol or normal cell or organism, exhibiting, for example, normaltraits. In yet another embodiment, a “suitable control” or “appropriatecontrol” is a predefined value, level, feature, characteristic,property, etc.

A “non-natural amino acid” (nnAA) is an amino acid that is not a memberof the canonical 20 amino acids normally encoded by the genetic code.Such nnAAs are organic compounds that have structures similar to anatural amino acid but have been modified structurally to mimic thestructure and reactivity of a natural amino acid. nnAAs can sharebackbone structures, and/or even the most side chain structures of oneor more natural amino acids, with the only difference(s) beingcontaining one or more modified groups in the molecule. nnAAs thusinclude, for example, amino acids or analogs of amino acids other thanthe 20 naturally-occurring amino acids and includeNε-(3-(4-iodophenyl)propanoyl)lysine andNε-(4-(4-iodophenyl)butanoyl)lysine.

The term “thioyl” refers to a divalent chemical functional group that isconventionally represented as a carbon atom having a double bond to asulfur atom.

A “non-sense” codon means a nucleotide triplet that in most organismsdoes not encode an amino acid. The term is synonymous with “stop codon”and “termination codon.” There are three stop codons, amber, ochre andopal. In RNA, they are respectively UAG, UAA, and UGA; in DNA, they arerespectively TAG, TAA, and TGA.

Compounds

In an aspect, compounds are disclosed herein that comprise albumintargeting groups. An “albumin targeting group,” “albumin targetingmolecule,” or “albumin targeting tag” is a small molecule that isincorporated into a second molecule, such as a polypeptide, such thatthe small molecule directs the second molecule to associate withalbumin, in vitro or preferably in vivo. Such association comprises abinding interaction between the albumin and the albumin targeting tag.In one embodiment, the albumin targeting group comprises an aryl orheteroaryl moiety and a thioyl moiety, such that the non-natural aminoacid (e.g., A-T, as described herein) comprises a thioamide moiety. In aparticular embodiment, the thioyl moiety is conjugated to the aryl orheteroaryl moiety.

Exemplary albumin targeting groups are shown in Scheme 1 below (DumelinC E, et al. Angew Chem, Int Ed. 2008; 47, 3196-201; WO2008053360). Othersuch albumin targeting tags are disclosed in Koehler, M F T, et al.(Bioorg. Med. Chem. Lett. 2002, 12:2883); and in Zobel, K., et al.(Bioorg. Med. Chem. Lett. 2003. 13:1513). Preferably, the albumin ishuman serum albumin.

To assess albumin binding, any assay designed to measure albumin bindingcan be used. Chromatographic binding assays are especially useful, suchas that described by Hage, D S, et al. J (Chromatogr B Biomed Sci Appl.2000, 739:39-54). Alternatively or in conjunction, binding affinity toalbumin can be determined by using surface plasmon resonance. In onemethod, a BIAcore 200 device (BIAcore Inc.; Piscataway, N.J.) is used incombination with a procedure by Lacy, S E, et al. (MAbs, 2015. 7:605-19)as modified herein. Briefly, human and mouse albumin are captured on aCM5 chip using amine coupling at 5000 resonance units. The candidatepolypeptides are injected at various concentrations (e.g., 0, 0.625,1.25, 5, and 10 μM) at a controlled flow rate (e.g., 20 μl/min). Thebound peptides are allowed to dissociate for a period of time (e.g., 5min) before matrix regeneration using 10 mM glycine, pH 3. The signalfrom an injection passing over an uncoupled cell is subtracted from thatof an immobilized cell to generate sensorgrams of the amount ofpolypeptide bound as a function of time. BIAcore kinetic evaluationsoftware (version 3.1) can be used to determine K_(D) from theassociation and dissociation rates using a one-to-one binding model.Alternatively or in conjunction, affinity to albumin can be determinedusing a direct assay, such as described by Nguyen, A, et al. (ProteinEng Des Sel. 2006, 19:291-7). In this method, albumin is immobilizedonto high protein-binding capacity polystyrene 96 well plates at apredetermined concentration, such as 2 mg/ml, for a period of time(e.g., overnight) at 4° C. Non-specific binding sites are blocked withbinding buffer, and radio-labelled candidate polypeptides are applied tothe plates for a period of time, such as 30 minutes, at 25° C. Unboundcandidate polypeptides are washed away, and bound candidate polypeptidesdetected using a radioactivity plate reader.

In another aspect, the polypeptides that are targeted to associate withalbumin via an albumin targeting molecule comprise at least one aminoacid that incorporates the albumin targeting molecule, thus creating annAA. Exemplified herein are the nnAAsNε-(4-(4-iodophenyl)butanoyl)lysine,Nε-(3-(4-iodophenyl)propanoyl)lysine and pharmaceutically acceptablesalts thereof (e.g., acid addition salts such as hydrochloride). See,e.g., the compounds of Scheme 2. Nε-(3-(4-iodophenyl)propanoyl)lysinebinds albumin poorly, while Nε-(4-(4-iodophenyl)butanoyl)lysine, havinga proper albumin binding tag, strongly associates with albumin.

The exemplified nnAAs 1 and 2 can be made from correspondingN-hydroxysuccinimide (NHS)-esters by standard conjugation chemistry,followed by deprotection with trifluoroacetic acid (TFA) andion-exchange to the chloride salts. Similarly, the exemplified nnAAs1^(S) and 2^(S) can be made by condensing protected lysine with thecorresponding thionacid derivatives of nitrobenzotriazole followed bydeprotection and ion-exchange (See, e.g., Thiopeptide Synthesis.Alpha-Amino Thioacid Derivatives of Nitrobenzotriazole as ThioacylatingAgents, M. Ashraf Shalaby, Christopher W. Grote, and Henry Rapoport, J.Org. Chem., 1996, 61, 9045-9048).

In some embodiments, the nnAAs are radiolabelled. For example,radioiodinated nnAA analogues comprising ¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I areshown in Scheme 3. The syntheses of radioiodinated non-natural aminoacids are performed analogously to the syntheses of non-natural aminoacids 1-4 (see, e.g., Example 1).

The radiolabel can be selected by one of skill in the art and willdepend in part on the type of preferred isotope, the purpose of theradiolabelled polypeptide, the half-life of the isotope, the cost of theisotope, and the facility with which to add the radiolabel to themolecule at a desired location. nnAAs can be purified afterradiolabelling using standard techniques.

Particularly with Nε-(4-(4-iodophenyl)butanoyl)lysine, it can beadvantageous for the surrounding residues to be modified. Thesurrounding residues are within two residues to each side of the nnAA.Such a “molecular context” can enhance the binding of the targetedmolecule to albumin. Amino acid substitutions can be accomplished byengineering a polynucleotide encoding the polypeptide of interest usingtechniques common in the art to introduce mutations to randomize thesesurrounding that encoding the nnAA. Methods include site-directedmutagenesis or PCR-mediated mutagenesis. Upon construction of thedesired mutant polynucleotides, the encoded proteins can be expressedand tested for enhanced binding to albumin, using such assays as aredescribed above.

Compositions

In some embodiments, herein disclosed are cells comprising apolynucleotide encoding a first polypeptide that when expressed by thecells are substituted with a radiolabelled non-natural amino acid at anonsense codon in the polynucleotide, creating a radiolabelled modifiedpolypeptide, wherein: the cells comprise a suppressor tRNA and itscognate tRNA synthetase that recognize the nonsense codon; thenon-natural amino acid is introduced into the first polypeptide by anonsense codon; and the radiolabelled non-natural amino acid isexogenously supplied.

In other aspects, disclosed herein are cells comprising a polynucleotideencoding a first polypeptide that when expressed by the cells aresubstituted with a non-natural amino acid comprising an albumintargeting group at a nonsense codon in the polynucleotide, creating amodified polypeptide, wherein: the cells comprise a suppressor tRNA andits cognate tRNA synthetase that recognize the nonsense codon; thenon-natural amino acid is introduced into the first polypeptide by anonsense codon; the non-natural amino acid is exogenously supplied; andthe modified polypeptide has enhanced binding to human serum albuminwhen compared to the first polypeptide.

In other aspects, disclosed herein are cells comprising a modifiedpolynucleotide encoding a modified polypeptide, a tRNA synthetase, and acognate tRNA, wherein the modified polynucleotide comprises one or morenonsense codons recognized by the tRNA synthetase and cognate tRNA,wherein the modified polypeptide comprises one or more non-natural aminoacids at positions encoded by the nonsense codons, and wherein thenon-natural amino acids comprise a moiety selected from the groupconsisting of: an albumin-targeting group, a radiolabel, and aradiolabelled albumin-targeting group.

In some aspects, disclosed herein are modified interleukin-1 receptorantagonist polypeptides comprising at least one non-natural amino acid,wherein the non-natural amino acid comprises a moiety selected from thegroup consisting of: an albumin-targeting group, a radiolabel, and aradiolabelled albumin-targeting group.

Methods

In some aspects, disclosed herein are methods of substituting a naturalamino acid in a first polypeptide with a non-natural amino acid toobtain a modified polypeptide, wherein the non-natural amino acidcomprises a moiety selected from the group consisting of: analbumin-targeting group, a radiolabel, and a radiolabelledalbumin-targeting group, comprising:

(a) selecting at least one amino acid residue in the first polypeptideto be substituted with the non-natural amino acid;

(b) selecting a polynucleotide encoding the first polypeptide;

(c) modifying the polynucleotide such that the amino acid residue to besubstituted is encoded by a nonsense codon;

(d) expressing the modified polynucleotide in a cell, wherein the cellis in the presence of the non-natural amino acid and expresses asuppressor tRNA and its cognate tRNA synthetase wherein the suppressortRNA recognizes the nonsense codon of step (c), and the cellincorporates the non-natural amino acid into the modified polypeptide atthe nonsense codon of step (c) during translation. The method canoptionally further include a purification step (e), wherein the modifiedpolypeptide is purified.

In other aspects, disclosed herein are methods of making a modifiedpolypeptide that binds to albumin, comprising:

(a) selecting at least one amino acid residue in a first polypeptide tobe substituted with Nε-(4-(4-iodophenyl)butanoyl)lysine;

(b) selecting a polynucleotide encoding the polypeptide;

(c) modifying the polynucleotide such that the amino acid residue to besubstituted is encoded by a nonsense codon;

(d) expressing the modified polynucleotide in a cell, wherein the cellis in the presence of the Nε-(4-(4-iodophenyl)butanoyl)lysine, or apharmaceutically acceptable salt thereof and expresses a suppressor tRNAand its cognate tRNA synthetase wherein the suppressor tRNA recognizesthe nonsense codon of step (c), and the cell incorporates theNε-(4-(4-iodophenyl)butanoyl)lysine into a modified polypeptide at thenonsense codon of step (c) during translation.

The method can optionally further include a purification step (e),wherein the modified polypeptide is purified.

In another aspect, disclosed herein are methods of engineering a cell,such as an Escherichia coli cell, to be resistant to radiation, whereinthe cell is used to introduce radiolabeled non-natural amino acids intopolypeptides, comprising mutating one or more proteins in the cell.

In another aspect, disclosed herein are methods of making a modifiedpolypeptide comprising

(a) selecting at least one amino acid residue in a first polypeptide tobe substituted with a non-natural amino acid, wherein the substitutedamino acid is solvent-accessible;

(b) selecting a polynucleotide encoding the first polypeptide;

(c) modifying the polynucleotide such that the amino acid residue to besubstituted is encoded by a nonsense codon; and

(d) expressing the modified polynucleotide in a cell, wherein the cellis in the presence of the non-natural amino acid and expresses asuppressor tRNA and its cognate tRNA synthetase wherein the suppressortRNA recognizes the nonsense codon of step (c), and the cellincorporates the non-natural amino acid into a modified polypeptide atthe nonsense codon of step (c) during translation.

The method can optionally further include a purification step (e),wherein the modified polypeptide is purified.

Selection of a Polypeptide

The selection of a polypeptide for targeting to albumin can bedetermined by one of skill in the art. In choosing such polypeptides,the polypeptide often has a therapeutic application, which functionwould benefit from binding to serum albumin to, for example, increasethe serum half-life of the therapeutic polypeptide in those embodimentsdirected to albumin targeting of polypeptides. General examples oftherapeutic polypeptides include, but are not limited to, antibodies,chimeric antibodies, monoclonal antibodies, single chain antibodies,Fab, Fab′, F(ab′)2, Fv, and scF, Fc fusions, anticoagulants, bloodfactors, bone morphogenetic proteins, engineered protein scaffolds,enzymes, growth factors, hormones, interferons, interleukins, andthrombolytics. Other examples of therapeutic peptides include: salmoncalcitonin; beta-interferon; gamma-interferon; veraglucerase-alpha;taliglucerase-alpha; glucarpidase (e.g., for treatment of methotrexatetoxicity); elosulfase-alpha (e.g., for treatment of Morquio syndrome);aldesleukin; anakinra; insulin lispro; uricase (e.g., for treatment ofgouty tophi); palifermin.

In some aspects, the polypeptide is interleukin-1 receptor antagonist(IL-1RA; SEQ ID NO:1, see Table 1; the mature form lacks the initiationmethionine: SEQ ID NO:2). Once modified with a nnAA having an albumintargeting group, such as the nnAA Nε-(4-(4-iodophenyl)butanoyl)lysine,then the modified IL-1RA will have enhanced binding to albumin than theunmodified IL-1RA. In additional aspects, the modified IL-1RA will haveenhanced pharmacokinetic properties, such as an increase in serumhalf-life when administered to a subject.

TABLE 1 Amino acid sequence of IL-1RA Met Arg Pro Ser Gly Arg Lys Ser Ser Lys Met Gln Ala Phe Arg Ile1               5                   10                  15 Trp Asp Val Asn Gln Lys Thr Phe Tyr Leu Arg Asn Asn Gln Leu Val            20                  25                  30 Ala Gly Tyr Leu Gln Gly Pro Asn Val Asn Leu Glu Glu Lys Ile Asp        35                  40                  45 Val Val Pro Ile Glu Pro His Ala Leu Phe Leu Gly Ile His Gly Gly    50                  55                  60 Lys Met Cys Leu Ser Cys Val Lys Ser Gly Asp Glu Thr Arg Leu Gln65                  70                  75                  80 Leu Glu Ala Val Asn Ile Thr Asp Leu Ser Glu Asn Arg Lys Gln Asp                85                  90                  95 Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser Gly Pro Thr Thr Ser Phe            100                105                  110 Glu Ser Ala Ala Cys Pro Gly Trp Phe Leu Cys Thr Ala Met Glu Ala        115                 120                 125 Asp Gln Pro Val Ser Leu Thr Asn Met Pro Asp Glu Gly Val Met Val    130                 135                 140 Thr Lys Phe Tyr Phe Gln Glu Asp Glu 145                 150Selection of Non-Natural Amino Acid (nnAA)

In some embodiments of the compositions and methods described herein,the non-natural amino acid comprises a moiety selected from the groupconsisting of: an albumin-targeting group, a radiolabel, and aradiolabelled albumin-targeting group.

In a particular embodiment, the non-natural amino acid comprises analbumin-targeting group. As described above, a non-natural amino acidcomprising an albumin-targeting group exhibits enhanced albumin bindingand pharmacokinetic properties relative to the corresponding naturalamino acid. Similarly, a polypeptide comprising a non-natural amino acidcomprising an albumin-targeting group exhibits enhanced albumin bindingand pharmacokinetic properties relative to an analogous polypeptidehaving a natural amino acid at the same position. For example, amodified polypeptide comprising the non-natural amino acid compound 1 ata particular position in the sequence will exhibit enhanced albuminbinding and pharmacokinetic properties relative to the correspondingunmodified polypeptide comprising a lysine at the particular position.

In another particular embodiment, the non-natural amino acid comprises aradiolabeled albumin-targeting group. In a preferred embodiment, theradiolabel is radioiodine (e.g., ¹²³I, ¹²⁴I, ¹²⁵I or ¹³¹I). Theincorporation of a radiolabel (e.g., a radioiodine) is useful forpurposes including investigations of polypeptide metabolism and tissuedistribution.

In some embodiments, the nnAA to be chosen for substitution can berepresented by the formula A-T, wherein A is an amino acid, and T is amoiety selected from the group consisting of: an albumin-targetinggroup, a radiolabel, and a radiolabelled albumin-targeting group. Insome embodiments, A is selected from the group consisting of lysine,ornithine, arginine, serine, threonine, asparagine and glutamine. In oneembodiment, T can be an albumin targeting group selected from Scheme 1or an albumin targeting group known to one of skill in the art, such asthose disclosed by Koehler, M F T, et al. (Bioorg. Med. Chem. Lett.2002, 12:2883) and in Zobel, K., et al. (Bioorg. Med. Chem. Lett. 2003.13:1513).

In a particular embodiment, A is lysine, and T is4-(4-iodophenyl)butyrate (i.e., A-T is compound 1, as shown in Scheme2). In another particular embodiment, the nnAA is selected from thespecies of Scheme 2 or Scheme 3.

Selection of Amino Acid Residue(s) to be Substituted with a nnAA

The selection of the amino acid residue(s) to be substituted isdetermined in part depending on the secondary and tertiary structure ofthe target polypeptide and the function of the target polypeptide. Theobjective, as understood by one of skill in the art, is to substitutewith a nnAA at permissive sites in the polypeptide, such that thesecondary and tertiary structure of the polypeptide is minimallydisturbed, if at all, and the function of the polypeptide is notsignificantly decreased. Preferred locations include those residues thatare on the surface of the target polypeptide, those that are solventaccessible, those that are away from any active or co-factor site (suchas in the case of enzymes) or binding site (such as in the case ofreceptor/ligand polypeptides).

Furthermore, the use of conservative amino acid substitutions can beuseful to minimally disturb the structure and function of thesubstituted polypeptide. A “conservative amino acid substitution” is onein which an amino acid residue is substituted by another amino acidresidue (including nnAA residues) having a side chain R group withsimilar chemical properties (e.g., charge or hydrophobicity). Ingeneral, a conservative amino acid substitution will not substantiallychange the functional properties of a protein. In cases where two ormore amino acid sequences differ from each other by conservativesubstitutions, the percent sequence identity or degree of similarity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well-known to thoseof skill in the art (Pearson, 1994, Methods Mol. Biol. 243:307-31).

Examples of groups of amino acids that have side chains with similarchemical properties include (1) aliphatic side chains: glycine, alanine,valine, leucine, and isoleucine; (2) aliphatic-hydroxyl side chains:serine and threonine; (3) amide-containing side chains: asparagine andglutamine; (4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; (5) basic side chains: lysine, arginine, and histidine; (6)acidic side chains: aspartic acid and glutamic acid; and (7)sulfur-containing side chains: cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.

Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al., 1992, Science 256:1443-1445. A “moderately conservative”replacement is any change having a nonnegative value in the PAM250log-likelihood matrix.

Sequence similarity for polypeptides is typically measured usingsequence analysis software. Protein analysis software matches similarsequences using measures of similarity assigned to varioussubstitutions, deletions and other modifications, including conservativeamino acid substitutions. For instance, Genetics Computer Group (GCGavailable from Genetics Computer Group, Inc.), also referred to as theWisconsin Package, is an integrated software package of over 130programs for accessing, analyzing and manipulating nucleotide andprotein sequences. GCG contains programs such as “Gap” and “Bestfit”which can be used with default parameters to determine sequencesimilarity, homology and/or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutein thereof. See,e.g., GCG version 6.1, version 7.0, version 9.1, and version 10.0.

Polypeptide sequences also can be compared using FASTA, a program inGCG, using default or recommended parameters. FASTA (e.g., FASTA2 andFASTA3) provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences (Pearson,1990, Methods Enzymol. 183:63-98; Pearson, 2000, Methods Mol. Biol.132:185-219). Another preferred algorithm when comparing a sequence ofthe invention to a database containing a large number of sequences fromdifferent organisms is the computer program BLAST, especially blastp ortblastn, using default parameters. See, e.g., Altschul et al., 1990, J.Mol. Biol. 215:403-410; Altschul et al., 1997, Nucleic Acids Res.25:3389-402.

As an example, in the case of human interleukin 1 receptor antagonist(IL-1RA; SEQ ID NO:1), where the nnAANε-(4-(4-iodophenyl)butanoyl)lysine is desired to be incorporated,permissive sites include Asn 135 (a conservative substitution), Lys71and Lys93 (both chosen in part because the selected nnAA is a derivativea lysine) because these sites are on the surface of the polypeptide, aresolvent accessible, and do not interfere with IL-1RA binding tointerleukin 1 receptor (IL-1R).

Selection of Polynucleotide

The polynucleotides selected for use with the methods disclosed hereinare those that encode the target polypeptides to be modified byincorporation of a nnAA. Such polynucleotides can be obtained in avariety of ways, including traditional cloning techniques andpolynucleotide synthesis. These approaches and others are well withinthe skill of a person in the art.

Polynucleotide Modification and Expression Systems

To incorporate the nnAA, one approach is to create a system wherein ausual non-sense codon, such as an amber codon, is used to encode for thennAA. Other codons that can be used include the opal and ochre nonsensecodons.

An expression system useful in the methods disclosed herein willcomprise: (1) a nnAA, (2) an expression system, (3) a tRNA synthetase,(4) the cognate tRNA of the tRNA synthetase, and (5) a polynucleotide tobe expressed that comprises the nonsense codon recognized by the tRNA.For example, U.S. Pat. No. 8,980,581 discloses the building and use ofexpression systems incorporating nnAAs.

The polynucleotide to be expressed can be modified by a number oftechniques to engineer the desired nonsense codon at the desiredlocation. For example, site-directed mutagenesis and PCR-mediatedmutagenesis can be used.

Host cells are genetically engineered with polynucleotide vectors, whichcan be, for example, a cloning vector or an expression vector. Thevector can be, for example, in the form of a plasmid, a bacterium, avirus, a naked polynucleotide, or a conjugated polynucleotide. Thevectors are introduced into cells and/or microorganisms by standardmethods. Transformation methods are well known in the art. For example,several well-known methods of introducing target nucleic acids intobacterial cells are available. These include: fusion of the recipientcells with bacterial protoplasts containing the DNA, electroporation,projectile bombardment, and infection with viral vectors.

Typical vectors contain transcription and translation terminators,transcription and translation initiation sequences, and promoters usefulfor regulation of the expression of the particular target nucleic acid.The vectors optionally comprise generic expression cassettes containingat least one independent terminator sequence, sequences permittingreplication of the cassette in eukaryotes, or prokaryotes, or both,(e.g., shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes, or both.

The expression system can be chosen such that the nonsense codon thesuppressor tRNA recognizes in the host cell is replaced in the nativegenome with other nonsense codons. In other aspects, the expressionsystem can be chosen such that the host cell lacks a release factor thatdecodes the nonsense codon as the termination signal of proteintranslation. In yet another aspect, the host cell both lacks a nonsensecodon, such as an amber codon, and lacks release factor1, which decodesthe amber codon as the termination signal.

Host cells can also be radiation resistant. In such aspects, the hostcell can be a bacterial cell; which, in some embodiments, is an E. colicell. In the case of an E. coli cell, the cell is engineered to comprisemutations in RecA (D276N), YfjK (A151D), and DnaB (P80H). In someaspects, the E. coli cell to be engineered is C321.ΔA (available asstrain 48998 from Addgene; Cambridge, Mass.).

Aminoacyl tRNA synthetases (AARSs) catalyze the aminoacylation reactionfor incorporation of amino acids into proteins via the correspondingtransfer RNA molecules. Precise manipulation of synthetase activity canalter the aminoacylation specificity to stably attach non-canonicalamino acids into the intended tRNA. Then, through codon-anticodoninteraction between message RNA (mRNA) and tRNA, the amino acid analogscan be delivered into a growing polypeptide chain. Thus, incorporationof non-natural amino acids into proteins relies on the manipulation ofamino acid specificity of AARS.

The AARS (or “synthetase”) can be a naturally occurring synthetasederived from an organism, whether the same (homologous) or different(heterologous), a mutated or modified synthetase, or a designedsynthetase.

The synthetase can recognize the desired nnAA selectively over availablerelated amino acids. For example, when the amino acid analog to be usedis structurally related to a naturally occurring amino acid (e.g.,lysine and Nε-(4-(4-iodophenyl)butanoyl)lysine), the synthetase shouldcharge the external mutant tRNA molecule with the desired nnAA with anefficiency at least substantially equivalent to that of, and morepreferably at least about twice, 3 times, 4 times, 5 times or more thanthat of the naturally occurring amino acid.

A synthetase can be obtained by a variety of techniques known to one ofskill in the art, including combinations of such techniques as, forexample, computational methods, selection (directed evolution) methods,and incorporation of synthetases from other organisms.

In certain embodiments, synthetases can be used or developed thatefficiently charge tRNA molecules that are not charged by synthetases ofthe host cell. For example, suitable pairs may be generally developedthrough modification of synthetases from organisms distinct from thehost cell. The synthetase can also be developed by selection proceduresor can be designed using computational techniques such as thosedescribed in Datta et al., J. Am. Chem. Soc. 124: 5652-5653, 2002, andin U.S. Pat. No. 7,139,665.

A strategy for generating an external mutant tRNA, modified or externalmutant AARS, or modified tRNA/AARS pair involves importing a tRNA and/orsynthetase from another organism into the translation system ofinterest, such as E. coli. The properties of the heterologous synthetasecandidate include, e.g., that it does not charge E. coli tRNA reasonablywell (preferably not at all), and the properties of the heterologoustRNA candidate include, e.g., that it is not acylated by E. colisynthetase to a reasonable extent (preferably not at all).

A similar approach involves the use of a heterologous synthetase as theexternal mutant synthetase and a mutant initiator tRNA of the sameorganism or a related organism as the modified tRNA.

The pairs and components of pairs desired above can be evolved togenerate external mutant tRNA and/or AARS that possess desiredcharacteristics, e.g., that can preferentially aminoacylate an O-tRNAwith a nnAA.

The modified tRNA and the modified AARS can be derived by mutation of anaturally occurring tRNA and AARS from a variety of organisms. Themodified tRNA and/or modified AARS are derived from at least oneorganism, where the organism is a prokaryotic organism, e.g.,Methanococcus jannaschii, Methanobacterium thermoautotrophicum,Halobacterium, E. coli, A. fulgidus, P. furiosus, P. horikoshii, A.pernix, and T. thermophilus. Optionally, the organism is a eukaryoticorganism, e.g., plants, algae, fungi, animals, insects, and protists.Optionally, the modified tRNA is derived by mutation of a naturallyoccurring tRNA from a first organism and the modified AARS is derived bymutation of a naturally occurring AARS from a second organism. Themodified tRNA and modified AARS can be derived from a mutated tRNA andmutated AARS. The modified AARS and/or modified tRNA from a firstorganism can be provided to a translational system of a second organism,which optionally has non-functional endogenous AARS and/or tRNA withrespect to the codons recognized by the modified tRNA or modified AARS.

The mutation or modification of an AARS to be used for incorporation ofa nnAA into a target polypeptide or protein can be performed by usingdirected mutagenesis once the desired contact amino acid residues havebeen identified. Identification of the contact amino acids can beperformed using any method that allows analysis of the structure of theAARS, including crystallographic analysis, computer modeling, nuclearmagnetic resonance (NMR) spectroscopy, library screening, or acombination of any of these or other methods.

A number of AARS molecules have been sequenced, which sequenceinformation provides guidance as to which amino acids are important forbinding the amino acid with which to charge the corresponding tRNA.

In certain embodiments, the AARS capable of charging a particularexternal mutant tRNA with a particular nnAA can be obtained bymutagenesis of the AARS to generate a library of candidates, followed byscreening and/or selection of the candidate AARS's capable of theirdesired function. Such external mutant AARSs and external mutant tRNAsmay be used for in vitro/in vivo production of desired proteins withmodified unnatural amino acids.

Methods for producing at least one recombinant external mutant AARScomprise: (a) generating a library of (optionally mutant) AARSs derivedfrom at least one AARS from a first organism, e.g., a eukaryoticorganism (such as a yeast), or a prokaryotic organism, such asMethanococcus jannaschii, Methanobacterium thermoautotrophicum,Halobacterium, E. coli, A. fulgidus, P. furiosus, P. horikoshii, A.pernix, and T. thermophilus; (b) selecting (and/or screening) thelibrary of AARSs (optionally mutant AARSs) for members that aminoacylatean external mutant tRNA in the presence of an unnatural amino acid and anatural amino acid, thereby providing a pool of active (optionallymutant) AARSs; and/or, (c) selecting (optionally through negativeselection) the pool for active AARSs (e.g., mutant AARSs) thatpreferentially aminoacylate the O-tRNA in the absence of the nnAA,thereby providing the at least one recombinant external mutantsynthetase, wherein the at least one recombinant external mutantsynthetase preferentially aminoacylates the external mutant tRNA withthe nnAA.

Libraries of mutant AARSs can be generated using various mutagenesistechniques known in the art. For example, the mutant AARSs can begenerated by site-specific mutations, random mutations, diversitygenerating recombination mutations, chimeric constructs, and by othermethods described herein or known in the art.

Selecting (and/or screening) the library of AARSs for members that areactive, e.g., that aminoacylate an external mutant tRNA in the presenceof a nnAA and a natural amino acid, includes: introducing a positiveselection or screening marker, e.g., an antibiotic resistance gene, orthe like, and the library of AARSs into a plurality of cells, whereinthe positive selection and/or screening marker comprises at least onecodon, which translation (optionally conditionally) depends on theability of a candidate AARSs to charge the external mutant tRNA (witheither a natural and/or a unnatural amino acid); growing the pluralityof cells in the presence of a selection agent; identifying cells thatsurvive (or show a specific response) in the presence of the selectionand/or screening agent by successfully translate the codon in thepositive selection or screening marker, thereby providing a subset ofpositively selected cells that contains the pool of active AARSs.

A cell-free in vitro system can be used to test the ability of theexternal mutant synthetase to charge the modified tRNA in a positivescreening. For example, the ability of the in vitro system to translatea positive screening gene, such as a fluorescent marker gene, may dependon the ability of the external mutant synthetase to charge modified tRNAto read through a codon of the marker gene.

Negatively selecting or screening the pool for active AARSs (optionallymutants) that preferentially aminoacylate the mutant tRNA in the absenceof the unnatural amino acid includes: introducing a negative selectionor screening marker with the pool of active AARSs from the positiveselection or screening into a plurality of translational system, whereinthe negative selection or screening marker comprises at least one codon(e.g., codon for a toxic marker gene, e.g., a ribonuclease barnasegene), which translation depends on the ability of a candidate AARS tocharge the external mutant tRNA (with a natural amino acid); and,identifying the translation system that shows a specific screeningresponse in a first media supplemented with the unnatural amino acid anda screening or selection agent, but fail to show the specific responsein a second media supplemented with the natural amino acid and theselection or screening agent, thereby providing surviving cells orscreened cells with the at least one recombinant AARS.

Methods for producing a recombinant external mutant tRNA include: (a)generating a library of mutant tRNAs derived from at least one tRNA,from a first organism; (b) selecting (e.g., negatively selecting) orscreening the library for (optionally mutant) tRNAs that areaminoacylated by an AARS (RS) from a second organism in the absence of aAARS from the first organism, thereby providing a pool of tRNAs(optionally mutant); and, (c) selecting or screening the pool of tRNAs(optionally mutant) for members that are aminoacylated by an introducedexternal mutant AARS, thereby providing at least one recombinant tRNA;wherein the at least one recombinant tRNA recognizes a non-natural aminoacid codon and is not efficiency recognized by the AARS from the secondorganism and is preferentially aminoacylated by the external mutantAARS.

AARS mutants can be generated from a library of mutants already havingthe desired activity. Mutant AARSs are selected, mutagenized (such as byerror-prone PCR) and subjected to positive and negative selection asgenerally described above. The objective of the positive selection is toidentify those mutants that can charge the tRNA with the nnAA; theobjective of the negative selection is to rid the identified AARSs fromthe positive selection that can charge the tRNA with endogenous aminoacids. For example, a positive screen is based on a bacterial cell's(such as E. coli) resistance to chloramphenicol, which is conferred bythe suppression of an amber mutation in the chloramphenicolacetyltransferase gene in the presence of the nnAA. An example of anegative screen is the use of a barnase gene having an amber mutationand is carried out in the absence of the nnAA. tRNA's that can becharged with endogenous amino acids allows for expression of the toxicbarnase gene and cells carrying such mutants die.

Suppressor tRNAs can also be optimized, desirable especially when thetRNA is derived from another organism than that derived for theexpression system. For example, the nucleotides in the T-stem and A-stemcan be randomized, as well as those nucleotides that form Watson-Crickbase pairing. tRNA mutants that are positively selected withchloramphenicol are then subjected to a screen to monitor efficiency ofthe tRNA to incorporate the nnAA, such as where green fluorescentprotein (GFP) is engineered with an amber nonsense codon; in thepresence of the nnAA and the mutant tRNA, the GFP is expressed, andsignal intensity can be used as a guide to understand the ability of thetRNA to incorporate the nnAA into the encoded protein.

In an embodiment, the AARS/tRNA pair is pyrrolysyl-tRNA synthetase(PylRS) and its cognate tRNA_(CUA) ^(Pyl) and wherein the nnAA isNε-(4-(4-iodophenyl)butanoyl)lysine. In an embodiment, PylRS is frommethanogenic archaea. PylRS and tRNA_(CUA) ^(Pyl) naturally incorporatepyrrolysine in response to the amber nonsense codon in some methanogenicarchaea (Blight, S K, et al. Nature, 2004: 333-5; Srinivasan, G, et al.Science, 2002, 296:1459-62). E. coli and animal cell endogenous AARSs donot recognize tRNA_(CUA) ^(Pyl) (Polycarpo, C, et al. PNAS 2004, 101:12450-4; Nozawa, K, et al. Nature, 2009, 457: 1163-7; Chen, P R, et al.Angew Chem Int Ed. 2009, 48:4052-5). Thus nnAA can be added to thegenetic code of mammalian cells and bacteria using the tRNA_(CUA)^(Pyl)/PylRS pair (Dumas, A., et al. Chemical Science. 2015, 6:50-69).In some embodiments, the PylRS is optimized forNε-(4-(4-iodophenyl)butanoyl)lysine. In other embodiments, thetRNA_(CUA) ^(Pyl) is optimized for NE-(4-(4-iodophenyl)butanoyl)lysine.In yet another embodiment, the tRNA_(CUA) ^(Pyl)/PylRS pair is optimizedfor Nε-(4-(4-iodophenyl)butanoyl)lysine.

Purification

The modified polypeptides can be purified from the cells and/or the cellmedia (if the modified polypeptide is secreted by the cells duringexpression) using well-known techniques. For example, the modifiedpolypeptide can be further modified to comprise an affinity tag,permitting easy purification of the modified polypeptide from theexpression system. Tags are typically placed at the polypeptide's N- orC-terminus, where they can be easily removed in subsequent processing.

A frequently used affinity tag is a multi-histidine tag (e.g., 6×His),which has an affinity towards nickel or cobalt ions. If nickel or cobaltions are immobilized on a solid carrier, such as a resin column, theHis-tagged polypeptides can be depleted from a cell lysate or cell mediaby running the lysate or media through the column. Such techniques arewell known in the art, and His-tag vectors are commercially available.

Other affinity tags can be used, which allow rapid removal of themodified polypeptide by immunoaffinity-based separation technique, suchas immunoaffinity chromatography. Exemplary tags include GreenFluorescent Protein (GFP), Glutathione-S-transferase (GST), and theFLAG-tag.

Simple immunoaffinity purification, without any tags, can also be used.

EXAMPLES

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of noncriticalparameters which can be changed or modified to yield essentially thesame results.

Example 1: Amino Acid Synthesis and Radiosynthesis

The two non-natural amino acids (nnAAs 1 and 2; FIG. 1) were synthesizedfrom the corresponding NHS-esters by standard conjugation chemistry(FIG. 1). We performed a chromatographic assay of HSA binding (Hage D S,Austin J. High-performance affinity chromatography and immobilized serumalbumin as probes for drug- and hormone-protein binding. J Chromatogr BBiomed Sci Appl. 2000; 739, 39-54) for these two tags (K_(d)=7.0 μM for1; K_(d)=204 μM for 2) and confirmed the significant difference in HSAaffinity observed previously (Dumelin C E, et al. A portable albuminbinder from a DNA-encoded chemical library. Angew Chem, Int Ed. 2008;47, 3196-201). To establish that radiolabeled 1 and 2 could be preparedon a timescale commensurate with protein biosynthesis, diaryliodoniumsalt precursors 5 and 6 (FIG. 1) were treated with no-carrier-added(n.c.a.) Na¹²⁴I (2 mCi) to afford ¹²⁴I-labeled 3 and 4 in excellentradiochemical yield with little to no hydrolysis of the activated ester.Rapid purification (ethyl acetate, silica sep-pak) provided sufficientlypure material for peptide labeling studies. These results demonstratethat high specific activity ¹²⁵I-labeled 1 and 2 can be prepared quickly(within 3 hours) and in sufficient radiochemical yield for same-daybiosynthesis of proteins, such as the IL-1RA mutant proteins.

Compounds 5 and 6 were prepared according to, or in analogy to, themethod of Example 11.

Example 2: Protein Synthesis Using 1 and 2

Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA_(CUA) ^(Pyl)naturally incorporate pyrrolysine (Pyl) in response to the ambernonsense codon in some methanogenic archaea (Blight S K, et al. Directcharging of tRNACUA with pyrrolysine in vitro and in vivo. Nature. 2004;431, 333-5; Srinivasan G, et al. Pyrrolysine Encoded by UAG in Archaea:Charging of a UAG-Decoding Specialized tRNA. Science. 2002; 296,1459-62). Previous work has shown that tRNA_(CUA) ^(Pyl) is notrecognized by endogenous aminoacyl-t-RNA synthetases (AARSs) in E. colior mammalian cells (Polycarpo C, et al. An aminoacyl-tRNA synthetasethat specifically activates pyrrolysine. Proc Natl Acad Sci USA. 2004;101, 12450-4; Nozawa K, et al. Pyrrolysyl-tRNA synthetase-tRNAPylstructure reveals the molecular basis of orthogonality. Nature. 2009;457, 1163-7; Chen P R, et al. A Facile System for Encoding UnnaturalAmino Acids in Mammalian Cells. Angew Chem Int Ed. 2009; 48, 4052-5),and that non-natural amino acids (nnAAs) can be added to the geneticcode of mammalian cells and bacteria using the tRNA_(CUA) ^(Pyl)/PylRSpair (Dumas A, et al. Designing logical codon reassignment—Expanding thechemistry in biology. Chemical Science. 2015; 6, 50-69). Moreimportantly, many of these nnAAs are structurally similar to 1 and 2.

Since many PylRS mutants have broad substrate specificity, we firstscreened only a small library of 22 previously evolved PylRS mutants fortheir ability to incorporate 1 and 2 into green fluorescence protein(GFP) in E. coli. The screening was based on the suppression of an ambernonsense codon at a permissive site in a GFP-encoding gene. Briefly, aplasmid, pLei-GFP_(UV)-N149UAG, which contains GFP_(UV)-149UAG and acopy of a tRNA_(CUA) ^(Pyl), was co-transformed into E. coli GeneHogsstrain with a plasmid containing a variant of PylRS mutant. Proteinexpression was carried out in control LB medium and in mediumsupplemented with 1 mM 1 or 2. After induction of GFP expression andcultivation (12 h), cells were collected, washed, and analyzed using afluorescence plate reader. (Incorporation efficiency scales withfluorescence.) As shown in FIG. 2, fluorescence analysis of E. colicultures showed that significantly more full-length GFP_(UV) proteinswere produced in the presence of 1 or 2 for the best hit from thelibrary. The fact that a small preliminary screen identified a mutantcapable of incorporating 1 into GFP efficiently (20 mg/L vs. 80-120 mg/Lfor wt) indicates that further PylRS optimization is highly likely toproduce an efficient expression system for proteins that incorporate(p-iodophenyl)alkanoyl nnAAs.

Example 3: Evolution and Optimization of Pyrrolysyl-tRNA Synthetase andits Cognate tRNA_(CUA) ^(Pyl) to Encode Homologous Non-Natural AminoAcids Derived from (Nε-(3-(4-Iodophenyl)propanoyl)lysine andNε-(4-(4-Iodophenyl)butanoyl)lysine) into IL-1RA Mutants in E. coli

Three approaches are used to achieve efficient production of proteinsincorporating the homologous nnAA pair (1 and 2).

Example 3A: Optimization of the Hit from the Initial Screen

The most promising hit from the initial screen (DizPKRs-Y349F, FIG. 2)serves as the basis for an additional mutant library, generated usingerror-prone PCR. A previously established selection system (Chen P R, etal. A Facile System for Encoding Unnatural Amino Acids in MammalianCells. Angew Chem Int Ed. 2009; 48, 4052-5) is used to identify PylRSmutants with increased activities. Briefly, the library is subjected toconsecutive rounds of negative selection and positive selection usingreporter genes containing an amber codon at a permissive position. Thepositive selection identifies functional PylRS mutants and is based oncells' resistance to chloramphenicol, which is conferred by thesuppression of an amber mutation in the chloramphenicolacetyltransferase gene in the presence of the nnAA. The negativeselection removes any PylRS mutants that can charge tRNA_(CUA) ^(Pyl)with endogenous amino acids; if tRNA_(CUA) ^(Pyl) is loaded with anendogenous amino acid, suppression of amber mutations in the toxicbarnase gene leads to cell death. The negative selection is carried outin the absence of nnAAs. To determine the efficiency and fidelity of 1and 2 incorporation by the evolved PylRS mutants in E. coli, we use thesame fluorescence-based assay featured in our preliminary study (Example2). In addition, the GFP protein is purified by affinity chromatographyand analyzed by tandem mass spectrometry.

Example 3B: Identification of New Hits by De Novo Selection UsingSeveral Large PylRS Mutant Libraries

Since the hit we identified from the initial screening was optimizedpreviously for incorporation of a different nnAA, it is unlikely to bethe best PylRS variant for the incorporation of 1 and 2. Formalselection using a structure-guided, directed evolution approach is used.Based on the structure of PylRS (Nozawa K, et al. Pyrrolysyl-tRNAsynthetase-tRNAPyl structure reveals the molecular basis oforthogonality. Nature. 2009; 457, 1163-7; Kavran J M, et al. Structureof pyrrolysyl-tRNA synthetase, an archaeal enzyme for genetic codeinnovation. Proceedings of the National Academy of Sciences. 2007; 104,11268-73) and molecular modeling, we have constructed several PylRSmutant libraries in which residues within the amino acid binding pocketof PylRS were randomized. Among these libraries, three of them arederived from Methanosarcina maize PylRS (Library 1: L305, Y306, L309,N346, and C348; Library 2: L305, Y306, L309, C348, and V401; Library 3:L305, Y306, L309, C348, and Y384) and three of them are derived fromMethanosarcina barkeri PylRS (Library 1: M241, A267, L270, Y271, andL274; Library 2: M265, L266, A267, C346, and M347; Library 3: L270,Y271, L274, V313, and V401). Our established standard selection processis applied.

Example 3C: tRNA Engineering to Improve Incorporation Efficiency of 1and 2

The suppressor tRNA_(CUA) ^(Pyl) derived from archaea is a criticalelement in amber nonsense suppression; it is engineered to accommodateE. coli translational machinery. We focus on the T-stem and the portionof the A-stem of tRNA that interact with EF-Tu. Previously, we showedthat the optimization of interaction between archaea Methanococcusjannaschii tRNA and E. coli EF-Tu led to up to 25-fold increase in ambersuppression efficiency in E. coli (Guo J, et al. Evolution of AmberSuppressor tRNAs for Efficient Bacterial Production of ProteinsContaining Nonnatural Amino Acids. Angew Chem Int Ed. 2009; 48,9148-51). It is likely that a similar tRNA engineering strategy wouldlead to improved amber suppression efficiency of the PylRS-tRNA_(CUA)^(Pyl) pair in E. coli. We construct tRNA libraries in which thenucleotides in the T-stem and A-stem is completely randomized. Thenucleotides that form Watson-Crick base pairing with the abovenucleotides is randomized as well. To identify tRNA mutants with highersuppression efficiency, our previously established selection system isused. The tRNA_(CUA) ^(Pyl) mutants that can survive in the presence ofthe highest concentration of chloramphenicol is selected for furtherevaluation using our GFP expression test (Guo J., et al, supra).

By means of the three complementary approaches outlined above, we obtainPylRS-tRNA_(CUA) ^(Pyl) pairs that display desirable incorporationefficiencies for 1 and 2.

Example 4: Synthesis of IL-1RA with 1 and 2

Based on the structure of interleukin-1 receptor (IL-1R) and theinterleukin-1 receptor antagonist (IL-1RA) complex (Schreuder H, et al.A new cytokine-receptor binding mode revealed by the crystal structureof the IL-1 receptor with an antagonist. Nature. 1997; 386, 194-200),three residues at permissive sites of IL-1RA were selected forsubstitution with 1 and 2. Two of the permissive sites are lysineresidues and the other one is an asparagine residue. All three arelocated on the surface of IL-1RA and do not interfere with its bindingto IL-1R (FIG. 3). The control and mutant proteins are expressed in E.coli in the presence of the evolved PylRS-tRNA_(CUA) ^(Pyl) pair and thenon-natural amino acid of interest (1 or 2), and purified as6×His-tagged proteins by affinity chromatography. The 6×His-tag isremoved by protease digestion subsequently, if necessary. The purity ofthe proteins is assessed by SDS-PAGE and the sequence is confirmed byMS-MS.

Example 5: Incorporation of Protein Surface Carboxylates into IL-1RaMutants and Assessment of Impact of Such Substitutions on In VitroBinding of Tagged IL-1RA Mutants to Human Serum Albumin and IL-1R

Our preliminary albumin binding results with 1 and its correspondingmethyl ester demonstrated the carboxylate group provided an increase inalbumin affinity. In their initial report, Dumelin and coworkerssuggested that the free carboxylate of 1 (D- or L-isomer) mimicked the5′-phosphate found in the DNA encoded library screen to enhance albuminaffinity (Dumelin C E, et al. A portable albumin binder from aDNA-encoded chemical library. Angew Chem, Int Ed. 2008; 47, 3196-201).Intriguingly, they found that the anionic group needed to be six or morecarbon atoms removed from the amide bond of the tag to achieve strongalbumin targeting. Given the rather severe structural constraints of theaminoacyl-t-RNA synthetases (AARS) used to introduce nnAAs, it wasthought far easier to introduce negatively charged amino acids near thesite of tag insertion using standard site-directed mutagenesis, ratherthan attempt to evolve an AARS to accept a branched, negatively chargednnAA. To test the feasibility of the “encoded charge” approach toenhanced albumin targeting, we prepare the four dipeptides of 1 with Gluand Asp (FIG. 4), and assess their albumin affinity using achromatographic binding assay described earlier (Hage D S, Austin J.High-performance affinity chromatography and immobilized serum albuminas probes for drug- and hormone-protein binding. J Chromatogr B BiomedSci Appl. 2000; 739, 39-54). The data from these studies allow us todetermine if albumin binding is sensitive to the position and identityof the negatively-charged carboxylate group; these data are used toinform and interpret the consequence of introducing a second mutantresidue (Glu or Asp) in the vicinity of the albumin-targeting tag inIL-1RA.

Contemporaneously with these chemical synthesis studies, we alsoconstruct a small IL-1RA mutant library in which the two positions oneither side of nnAA 1 are randomized. A subsequent chromatographic HSAbinding assay contrasting these compounds with “control” 1-tagged IL-1RAmutants and wt IL-1RA is used to determine the generality andeffectiveness of a protein surface charge in enhancing HSA affinity.Once the three best IL-1RA double mutants (one each selected fromIL-1RA-Lys71-1, IL-1RA-Lys93-1, and IL-1RA-Asn135-1, positions referringto those of SEQ ID NO:1) are identified, these proteins and their2-labeled homologs can be produced in quantity and subjected to detailedprotein receptor studies. If a second mutation is indeed necessary foreffective albumin binding, the perturbation introduced into the proteinstructure (2 amino acids instead of one) is still quite small.

Example 6: HSA Affinity Determination by Surface Plasmon Resonance

The binding affinities between IL-1RA and IL-1RA mutants and albumin areobtained using a BIAcore 2000 (BIAcore Inc., Piscataway, N.J.) using amodified procedure of Lacy (Lacy S E, et al. Generation andcharacterization of ABT-981, a dual variable domain immunoglobulin(DVD-Ig™) molecule that specifically and potently neutralizes both IL-1αand IL-1β. MAbs. 2015; 7, 605-19). Human and mouse albumin is capturedon a CM5 chip using amine coupling at 5000 resonance units. IL-1RA andIL-1RAmutants at 0, 0.625, 1.25, 2.5, 5, and 10 μm is injected at a flowrate of 20 μl/min for 30 s. The bound peptides are allowed to dissociatefor 5 min before matrix regeneration using 10 mm glycine, pH 3. Thesignal from an injection passing over an uncoupled cell is subtractedfrom that of an immobilized cell to generate sensorgrams of the amountof peptide bound as a function of time. The running buffer, PBScontaining 0.05% Tween 20, is used for all sample dilutions. BIAcorekinetic evaluation software (version 3.1) is used to determine K_(D)from the association and dissociation rates using a one-to-one bindingmodel.

Example 7: HSA Affinity Determination Direct Binding Assay

Albumin binding of radiolabeled IL-1RA and IL-1RA mutants (see below forthe synthesis of the mutant proteins) is assessed using a modificationto a procedure previously described by Nguyen (Nguyen A, et al. Thepharmacokinetics of an albumin-binding Fab (AB.Fab) can be modulated asa function of affinity for albumin. Protein Eng Des Sel. 2006; 19,291-7). Briefly, mouse and human albumin (Sigma; St. Louis, Mo.) areimmobilized onto NUNC Maxisorp® 96-well plates at 2 mg/ml overnight at4° C. The plates are blocked with binding buffer (PBS, 0.5% ovalbuminand 0.05% Tween-20) for 1 h at 25° C. IL-1RA mutants are seriallydiluted in binding buffer and added at 100 μl per well to theimmobilized albumin for 30 m at 25° C. Unbound IL-1RA mutants areremoved by washing wells with 0.05% PBS/Tween-20 and bound IL-1RAmutants are detected using a radioactivity plate reader. Alternatively,radiolabeled IL-1RA and IL-1RA mutants are assessed for albumin bindingusing ultrafiltration as described by Mueller (Muller C, et al. DOTAconjugate with an albumin-binding entity enables the first folicacid-targeted ¹⁷⁷Lu-radionuclide tumor therapy in mice. J Nucl Med.2013; 54, 124-31).

Example 8: Receptor Binding Studies with IL-1RA and IL-1RA Mutants

Affinity measurements are performed using IL-1R expressing cells(Steinkasserer A, et al. Human interleukin-1 receptor antagonist. Highyield expression in E. coli and examination of cysteine residues. FEBSLett. 1992; 310, 63-5; van der Laken C J, et al. Different behavior ofradioiodinated human recombinant interleukin-1 and its receptorantagonist in an animal model of infection. Eur J Nucl Med. 1996; 23,1531-5). The mouse helper T lymphocyte cell line D10.G4.1 (ATCC;Manassas, Va.) or EL-4-6.1 (Ludwig Institute for Cancer Research,Switzerland) cells are suspended at 10⁷ cells/ml in binding medium (RPMI1640, 5% FCS, 20 mM HEPES pH 7.2), and 50 μl of cell suspension wasadded to each well of a 96-well filtration plate (Millipore). To eachwell 50 microliters of ¹²⁵I-IL-1RA mutants at a final concentration of50 pM is added. Unlabeled human IL-1RA (IL-1RA and IL-1RA mutantsprepared in this work) are serially diluted in binding medium, and 50 μlaliquots of unlabeled ligand are added to each well. Receptor binding isallowed to proceed to equilibrium at 4° C. The plates are washed inbinding medium by vacuum filtration, and the filters subsequentlycounted for bound radioactivity. Statistical analysis is performed usingthe one-way analysis of variance (ANOVA).

Example 9: Preparation for Analyzing by SPECT and Classical Methods theBiodistributions of ¹²⁵I-Labeled Albumin Targeted and Control IL-1RAProteins in Normal Mice

A more direct, one-step approach to simplify the purification andisolation of ¹²⁵I-radiolabeled products is explored (FIG. 5). Given thatthis approach has been successfully applied to the synthesis of asimilarly protonated amine under acidic conditions (¹²⁵I-mIBG), nodifficulties with the second route are anticipated.

These radio-iodinated amino acids are used in the first ever proteinbacterial biosynthesis of radio-iodinated proteins for imaging. Thedrive to improve the efficiency of mutant IL-1RA protein production canbe understood after consideration of the expected ¹²⁵I-labeled IL-1RAprotein yields. Table 2 shows the amount of labeled protein activityexpected (per mL of medium) for no-carrier-added ¹²⁵I-labeled 2 atdifferent concentrations. While a 1% yield of protein from nnAAs isroutinely obtained and is sufficient for mouse bio-distribution andimaging studies (50 μCi of IL-1RA from 5 mCi of nnAA), we have set abenchmark of >5% incorporation to reduce the amount of activity thatneeds to be handled and to reduce the impact of any radiolysis upon thebacterial cultures. Given the relatively small amounts of radioactivityused here and the low energy gamma emission of ¹²⁵I (35.5 keV), it seemsunlikely that radiolysis is a problem.

TABLE 2 Amount of labeled protein activity expected for no-carrier-added¹²⁵I-labeled 2 at different concentrations nnAA nnAA Activity ProteinActivity Concentration (mCi/ml) (μCi/ml) Yield @ 40 mg/liter   1 mM2.125 0.23% 4.8875 0.5 mM 1.0625 0.46% 4.8875 0.1 mM 0.2125 2.30% 4.88750.05 mM  0.10625 4.60% 4.8875 Yield @ 100 mg/liter   1 mM 2.125 0.58%12.21875 0.5 mM 1.0625 1.15% 12.21875 0.1 mM 0.2125 5.75% 12.21875 0.05mM  0.10625 11.50%  12.21875

Example 10: Imaging and Biodistribution Studies

Tissue distribution in wild type BALB/C mice is performed for¹²⁵I-labeled IL-1RA (native) and two IL-1RA mutants labeled with 1 andtwo mutants labeled with 2 that demonstrate the highest IL-1R bindingaffinities. Tissue uptake is analyzed after administering, via the tailvein, a bolus injection of 10 μCi/mouse in a constant volume of 0.05 mL.Groups of 5 animals are euthanized by asphyxiation with carbon dioxideat 1, 4, 12, 24, 72 and 96 hours post injection. Tissues (tumor, blood,heart, liver, lungs, spleen, large and small intestine, stomach,kidneys, skeletal muscle, bone, adipose, testes, brain and tumor) aredissected, excised, weighed wet, transferred to plastic tubes andcounted in an automated γ-counter. Tissue time-radioactivity levels areexpressed as % injected dose per gram tissue (% ID/g) and % injecteddose per organ (% DPO).

Imaging studies are performed for ¹²⁵I-labeled IL-1RA (native) and theIL-1RA mutant tagged with 1 that shows the greatest blood retention asdetermined by the biodistribution studies above. The same mutant taggedwith 2 serves as a second control. Imaging studies are carried out usingthe Inveon Trimodality imaging system (Siemens; Deerfield, Ill.) inmicroSPECT mode. Animals are injected with 50 μCi/mouse of labeledIL-1RA (wt or mutant). Groups of 3 animals are injected per compoundstudied and imaging takes place at 1, 24, 72 and 96 h post injection.After the last time point, animals are euthanized by asphyxiation withcarbon dioxide. Tissues (tumor, blood, heart, liver, lungs, spleen,large and small intestine, stomach, kidneys, skeletal muscle, bone,adipose, testes, brain and tumor) are dissected, excised, weighed wet,transferred to plastic tubes and counted in an automated γ-counter.Tissue time-radioactivity levels are expressed as % injected dose pergram tissue (% ID/g) and % injected dose per organ (% DPO).

Example 11: Synthesis of Phenylpropanoic Acid Succinimidyl EsterDiaryliodonium Salt (6)

(1) 3-(4′-Iodophenyl)propanoic acid

A mixture of 3-phenylpropanoic acid (6.00 g, 40.0 mmol), H₅IO₆ (2.00 g,8.60 mmol), iodine (4.06 g, 16.0 mmol) and 98% H₂SO₄ (1.2 mL) in water(8 mL) and acetic acid (40 mL) was heated at 67° C. for 17 h. Thereaction mixture was cooled and quenched with water (100 mL). The crudeproduct was then filtered, washed with water and hexane. The pureproduct (6.2 g, 56%) was obtained by recrystallization from toluene.White solid. ¹H NMR (400 MHz, CDCl₃) δ 7.62 (d, J=8.4 Hz, 2H), 6.97 (d,J=8.0 Hz, 2H), 2.91 (t, J=7.6 Hz, 2H), 2.67 (t, J=7.6 Hz, 2H).

(2) 3-(4-Iodophenyl)propanoic acid succinimidyl ester

3-(4′-Iodophenyl)propanoic acid (10 mmol, 2.76 g, 1.0 eq.) andN-hydroxysuccinimide (15 mmol, 1.73 g, 1.5 eq) were dissolved inanhydrous CH₂Cl₂ (40 mL). The mixture was cooled to 0° C., andN,N′-dicyclohexylcarbodiimide (DCC, 15 mmol, 3.09 g, 1.5 eq) dissolvedin 20 mL CH₂Cl₂ was added drop-wise slowly. The mixture was stirredovernight at room temperature. The N,N′-dicyclohexylurea was filteredout, the residue was washed with CH₂Cl₂, and the filtrate was evaporatedto dryness purified by column chromatography and recrystallization withiso-propanol or toluene/hexane to afford succinimide as white solid(3.50 g, 94%). ¹HNMR (400 MHz, CD₃CN) δ 7.66 (d, J=8.4 Hz, 2H), 7.08 (d,J=8.4 Hz, 2H), 2.95 (A₂B₂, t, 4H), 2.75 (s, 4H). ¹³CNMR (100 MHz, CD₃CN)δ 168.4, 166.7, 138.1, 135.9, 129.2, 89.5, 30.1, 27.8, 23.8; HRMS (ESI)Calcd for C₁₃H₁₂NO₄INa (M+Na)+: 395.9709; Found: 395.9706.

(3)[4-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)phenyl]-(4′-methoxyphenyl)iodoniumtriflate (6)

In a N₂ charged glovebox, a solution of TMSOAc (13.0 mmol, 1.72 g, 2.6eq.) in 20 mL dry CH₃CN was added drop-wise to a solution ofSelectfluor™ (6.5 mmol, 2.30 g, 1.3 eq.) in 20 mL dry CH₃CN. Theresulting colorless mixture was then added drop-wise to a solution of(5.0 mmol, 1.87 g, 1.0 eq.) of 3-(4-Iodophenyl)propanoic acidsuccinimidyl ester in 20 mL dry CD₃CN. After being stirred at roomtemperature for 17 h, trifluoroborate salt (1.07 g, 5 mmol, 1.0 equiv.)was added to the solution followed by a solution of TMSOTf (1.00 g, 4.5mmol, 0.9 eq.) in 20.0 mL of dry CH₃CN was added drop-wise and themixture was allowed to stand at room temperature for 30 min. After thesolvent acetonitrile was removed, 100 mL of deionized water was addedand the mixture was extracted (30 mL×3) with CH₂CH₂. The combinedorganic layers were washed with water (50 mL×1) and the obtained waterlayers were extracted (50 mL×2) with CH₂CH₂ again. The combined organicextracts were dried over sodium sulfate, filtered, and the solvent wasremoved by rotary evaporation. The residue was dissolved in 1.0 mL CH₃CNand added drop-wise to 200 mL of MTBE to precipitate the diaryliodoniumtriflate product. This solid was dissolved in 1 mL acetonitrile/water(9:1 by volume) solution and slowly passed down an Amberlite IRA-400 ionexchange column (triflate counter ion). After removal of the solventsunder reduced pressure, the purified iodonium triflate product (2.49 g,79%) of was obtained as a colorless solid. ¹H NMR (400 MHz, CD₃CN) δ8.01 (d, J=9.2 Hz, 2H), 7.98 (d, J=8.4 Hz, 2H), 7.44 (d, J=8.4 Hz, 2H),7.05 (d, J=9.2 Hz, 2H), 3.84 (s, 3H), 3.07 (t, J=6.8 Hz, 2H), 2.97 (t,J=6.8 Hz, 2H), 2.74 (s, 4H); ¹³C NMR (75 MHz, CD₃CN) δ 170.0, 168.2,163.3, 145.4, 137.6, 135.1, 132.5, 118.1, 111.6, 101.6, 55.7, 31.3,29.5, 25.4; ¹⁹F NMR (CD₃CN, 282 MHz): d −79.2 (s, 3 F). HRMS (ESI) Calcdfor C₂₀H₁₉NO₅I (M-OTf)+: 480.0308; Found: 480.0310.

Example 12: Radioiodination of Compound (6)

Aqueous Na¹²⁴ I was dissolved in 0.1M NaOH. 1 μL of the Na¹²⁴I solution(approximately 1 mCi) was added to a first reaction vial along with 1 μLof 1.0 M AcOH to afford an acidic, slightly buffered solution. Theinitial activity was recorded for later calculations. Because the volumeof water was so small, initial drying of the Na¹²⁴I solution was notrequired.

5 mg of the diaryliodonium precursor[4-(3-((2,5-dioxopyrrolidin-1-yl)oxy)-3-oxopropyl)phenyl]-(4′-methoxyphenyl)iodoniumtriflate was dissolved in 400 μL of CH₃CN. The mixture was allowed tostand for 10 minutes to make certain all of the crystalline compound haddissolved. The diaryliodonium precursor was added to the first reactionvial and then the solution was evaporated with a stream of dry argon at90° C. After the solvent was removed completely, 125 μL of CH₃CN wasadded (with shaking or stirring) to dissolve the salts. Toluene (125 μL)was added and the solution was heated at 90° C. for 30 minutes. SilicaTLC (100% ethyl acetate) was performed to determine the labelingefficiency. The reaction mixture was purified by passing it through asilica sep-pak and the crude product was purified by reverse phase HPLCto afford the desired product in 94% yield.

Example 13: Synthesis of Thioamide Compound

(1) 4-(4′-Iodophenyl)butanoic acid

A mixture of 4-phenylbutanoic acid (20.0 g, 121.8 mmol), H₅IO₆ (5.56 g,24.4 mmol), iodine (13.30 g, 52.4 mmol), 10 M H₂SO₄ (5.0 mL), water (36mL) and acetic acid (166 mL) was heated at 70° C. for 19 h. The reactionmixture was cooled and evaporated to dryness. The residue was dissolvedin EtOAc (300 mL) and washed with aqueous Na₂S₂O₃ (2×200 mL), brine(2×200 mL), dried over Na₂SO₄, filtered, and evaporated to leave ayellow solid. The crude product was precipitated from EtOAc/hexane at 0°C. to afford product as light yellow solid (15.0 g, 42%). ¹H NMR (400MHz, CDCl₃) δ 11.0 (brs, 1H), 7.61 (d, J=8.4 Hz, 2H), 6.95 (d, J=8.0 Hz,2H), 2.63 (t, J=7.6 Hz, 2H), 2.38 (t, J=7.6 Hz, 2H).

(2) N-(2-amino-5-nitrophenyl)-4-(4-iodophenyl)butanamide

4-(4′-Iodophenyl)butanoic acid (11.60 g, 40 mmol) was dissolved in THF(200 mL), and N-methylmorpholine (NMM) (8.8 mL, 80 mmol, 2.0 equiv) wasadded at −20° C. under N₂. Isobutyl chloroformate (5.2 mL, 40 mmol, 1.0equiv) was added dropwise and the reaction mixture was stirred for 30min. A solution of 4-nitro-1,2-phenylenediamine (6.12 g, 40 mmol, 1.0equiv) in THF (100 mL) was added, and the mixture stirred for a further1.5 h at −20° C. and 15 h at 23° C. The precipitate was filtered off,and the filtrate was evaporated to dryness. The residue was dissolved inEtOAc (300 mL) and washed with aqueous solutions of 1 M NaH₂PO₄ (2×100mL), saturated brine (2×100 mL), saturated NaHCO₃ (2×100 mL), andsaturated NaCl (2×100 mL). The EtOAc solution was dried over Na₂SO₄, andevaporated to dryness. The crude product was sonicated in EtOAc until itsolidified. The remaining solid was filtered and dried in vacuo to yieldthe titled compound (10.77 g, 63%) as a yellow-brown solid.

(3) N-(2-Amino-5-nitrophenyl)-4-(4-iodophenyl)butanethioamide

P₂S₅ (4.44 g, 20 mmol, 1.0 equiv) was added to a suspension of Na₂CO₃(1.08 g, 10 mmol, 0.5 equiv) in THF (200 mL) at 23° C. under a flow ofN₂. After 1 h, the mixture was cooled to 0° C. andN-(2-amino-5-nitrophenyl)-4-(4-iodophenyl)butanamide (8.50 g, 20 mmol)in THF (100 mL) was added dropwise. The resulting mixture stirred for 2h at 0° C. and for 1 h at 23° C. The solvent was evaporated and theresidue was dissolved in EtOAc (200 mL), washed with 5% aqueous NaHCO₃(2×100 mL). The organic phase was separated and the aqueous phase wasextracted with EtOAc (100 mL). The combined organic phases were driedover Na₂SO₄, filtered and evaporated to dryness. The material wasdissolved in EtOAc, sonicated, filtered, and dried in vacuo to yield thetitled compound (6.89 g, 76%) as a yellow solid.

(4)4-(4-Iodophenyl)-1-(6-nitro-1H-benzo[d][1,2,3]triazol-1-yl)butane-1-thione

N-(2-Amino-5-nitrophenyl)-4-(4-iodophenyl)butanethioamide (5.68 g, 12.8mmol) was dissolved in glacial acetic acid (diluted with 5% water, 300mL) by gentle warming at 40° C. The solution was cooled to 0° C. andNaNO₂ (1.32 g, 19.2 mmol, 1.5 eq) was added in portions over 20 min withstirring. After 30 min, the precipitated product was filtered, washedwith water and the filtrate was extracted with EtOAc (2×150 mL). Thecombined organic phases were washed successively with H₂O (3×100 mL),saturated NaHCO₃ (2×100 mL), and brine (2×100 mL), was dried, and thenwas evaporated to dryness. The obtained solid was sonicated in a smallamount of EtOAc (5 mL). The EtOAc was decanted away and the remainingsolid was filtered. The product (yellow solid, 3.23 g, 56%) was combinedand dried in vacuo.

(5) N²-(tert-butoxycarbonyl)-N⁶-(4-(4-iodophenyl)butanethioyl)-L-lysine

A cooled solution (0° C.) of the thioacylating reagent(N-(2-amino-5-nitrophenyl)-4-(4-iodophenyl)butanethioamide, 5 mmol, 2.26g) in 75 mL of THF was treated, dropwise, with a solution of Boc-Lys-OH(5 mmol, 1.23 g) and triethylamine in 15 mL of THF and 3.0 mL of H₂Oover the course of 1 h. After the addition was complete, the mixture wasallowed to stir overnight at room temperature. The mixture was extractedwith EtOAc, and the organic layer was dried over Na₂SO₄, and evaporated.The residue was purified by silica gel flash chromatography(hexane/EtOAc, 1/1, followed by MeOH) to afford the title compound (1.18g) in 44% yield.

(6) N⁶-(4-(4-iodophenyl)butanethioyl)-L-lysine, trifluoroacetate salt

Trifluoroacetic acid (2.0 mL) was added to a solution ofN²-(tert-butoxycarbonyl)-N⁶-(4-(4-iodophenyl)butanethioyl)-L-lysine(1.05 g, 2 mmol) in dry DCM (2 mL), and the reaction mixture was allowedto stir for 4 h at room temperature. The solvents were evaporated underreduced pressure, and dried under vacuum overnight. The residue wasdissolved ethyl acetate (10.0 mL) and the solution was allowed to standundisturbed for 12 h. The precipitated was filtered and dried in vacuumto give the titled modified lysine derivative (0.97 g, 91%) as anoff-white solid.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Where any concept(s) orelement(s) of the invention is separately presented for convenience, itis understood that the combination of any such separately presentedconcept(s) or element(s), as necessary, is also encompassed by theinvention. Such equivalents are intended to be encompassed by theclaims.

The contents of the patents and references cited throughout thisspecification are hereby incorporated by reference in their entireties.

We claim:
 1. A method of substituting a natural amino acid in a firstpolypeptide with a non-natural amino acid to obtain a modifiedpolypeptide, wherein the non-natural amino acid comprises a moietyselected from the group consisting of: an albumin-targeting group and aradiolabelled albumin-targeting group, comprising: (a) selecting atleast one amino acid residue in the first polypeptide to be substitutedwith the non-natural amino acid; (b) selecting a polynucleotide encodingthe first polypeptide; (c) modifying the polynucleotide such that theamino acid residue to be substituted is encoded by a nonsense codon; (d)expressing the modified polynucleotide in a cell, wherein the cell is inthe presence of the non-natural amino acid and expresses a suppressortRNA and its cognate tRNA synthetase wherein the suppressor tRNArecognizes the nonsense codon of step (c), and the cell incorporates thenon-natural amino acid into the modified polypeptide at the nonsensecodon of step (c) during translation, wherein the albumin-targetinggroup or radiolabelled albumin-targeting group is selected from thegroup consisting of:


2. The method of claim 1, wherein the non-natural amino acid isNε-(4-(4-iodophenyl)butanoyl)lysine, or a pharmaceutically acceptablesalt thereof.
 3. The method of claim 1, wherein the non-natural aminoacid is Nε-(4-(4-iodophenyl)butanethioyl) lysine, or a pharmaceuticallyacceptable salt thereof.
 4. The method of claim 1, further comprisingstep (e), purifying the modified polypeptide.
 5. The method of claim 1,wherein the radiolabel is ¹²³I, ¹²⁴I, ¹²⁵I, or ¹³¹I.
 6. The method ofclaim 1, wherein the non-naturally occurring amino acid is selected fromthe group consisting of: